The invention concerns a system to determine the concentration of a substance mixed with a fluorophor; it also relates to its method of implementation.
The invention finds applications in numerous areas, in particular in the medical field for genome sequencing, the identification of mutations responsible for genetic diseases, the development of new medicinal products, etc.
Fluorescence (or short-lived photoluminescence) is a selective chemical analytical technique often used by persons skilled in the art to determine the concentration of a substance. The substance to be examined is mixed with a fluorophor. The fluorophor molecule, when excited at a wavelength xcex, has the property of re-emitting light with a spectrum whose maximum is greater than the emitted wavelength (xcexxe2x80x2 greater than xcex; STOKES"" law). As soon as excitation is stopped, fluorescence decreases exponential fashion : It=Io*exe2x88x92kt. Therefore, to observe a xe2x80x9cremanentxe2x80x9d fluorescence phenomenon, excitation needs to be maintained.
Also, it is known that not all the energy stored by the molecule is released in fluorescence form. The molecule maintains some of the energy in the form of vibration. This excess energy is dispelled by non-radiating processes called vibration relaxation mechanisms.
It is therefore possible to determine the fluorescence yield xcfx81, which is the ratio between the number of emitted photons and the number of absorbed photons; this yield is xcfx81=If/Ia.
It is shown that If=xcfx81*Io*xcex5*l*C in which Io is the exciting intensity, l is the thickness of the substance, C is the molar concentration and xcex5 is the molar absorption coefficient of the substance.
On the other hand, if the concentration increase is too great, fluorescence is no longer proportional to concentration as the solution absorbs quicker than its fluoresces. In this case the limits of the xe2x80x9clinear zonexe2x80x9d are exceeded.
If a CCD camera is positioned to form the fluorescence image, it can be said as a first approximation that the illumination received by the camera is proportional to If, fluorescence being isotropic in space; the situation is then E=xcfx80*L*dxcexa3/dS (LAMBERT""s law), in which E is the illumination, L is the luminance (constant for isotopic emission and proportional to excitation intensity), dxcexa3 is the emitter surface, and dS is the CCD pixel surface. However, the video signal leaving the camera is given by the ratio:Vos=q*xcex7*Nph*G/Cl, in which q is the electric charge, Nph is the number of incident photons, G is the gain, Cl is outgoing capacity and xcex7 is the quantum yield.
If the CCD camera is not saturated, illumination is proportional to integration time and to the number of photons received per time unit. It can then be written that Vos is proportional to the intensity of fluorescence If and integration time T, hence to the intensity of excitation Io, to integration time T, and to the molar concentration, the other parameters being fixed and dependent upon the instruments used; i.e.: Vos=k*Io*T*C*.
This formula is true if, and only if, work is conducted in a linear zone, that is to say if the molar concentration is not too high, if the excitation intensity does not cause absorption which would mask fluorescence, and if emission intensity and integration time do not saturate the CCD camera.
Therefore if it is desired to quantify fluorescence, it is essential to calibrate the experiment having the possibility of acting on two factors; integration time and the intensity of the excitation laser. This calibration may be made for example using contacts coated with a substance of known concentration. With a camera cooled to a very low noise level, it is possible to imagine that a resolution of 10 bits can be obtained, that is to say dynamics of 1000 or even more.
This fluorescence technique is used in particular for the identification and titration of the constituents of a DNA mixture applied to the surface of nucleic acid chip, called a DNA chip. The titration of the constituents of this mixture is made by using a DNA chip reader.
There currently exist numerous DNA chip readers, each adapted to a particular type of chip.
Documents U.S. Pat. No. 5,578,832, WO-97 4361 and U.S. Pat. No. 5,646,411 for example describe reading systems adapted to the chip produced by AFFYMETRIX, called the GENECHIP(copyright). Each of these systems consists of a scanning microscope used in confocal or non-confocal mode for point-by-point analysis of a surface area of approximately 10xc3x9710 mm in steps of 10 to 50 xcexcm.
Document U.S. Pat. No. 5,585,639 describes a linear strip reading system which provides sequential reading and reconstruction of a 2D image of chip fluorescence. This system is also adapted to the reading of GENECHIP(copyright) chips.
The size of the GENECHIP(copyright) is relatively large however for a chip, that is to say approximately 10xc3x9710 mm which is a disadvantage for some applications. Also, these chips comprise a high number of contacts, contiguous in both dimensions, which may make them difficult to localise. Finally, several contacts have to be read in order to achieve a biologically significant measurement.
The invention precisely discloses a reader that is adapted to another type of DNA chip, in particular to MICAM(copyright) chips which are of smaller size (1xc3x972 mm2) with less numerous contacts (100 to 1000) which are well spaced out (100 xcexcm). With this structure each contact is able to carry a well defined sequence that is perfectly well localised.
In other words, the invention concerns a system for determining the concentrations of the components of a mixed substance, for example containing DNA, applied to the surface of a MICAM(copyright) chip.
More precisely, the invention provides a system for determining the concentration of a substance mixed with a fluorophor and contained in one or more contacts of a matrix of conductor or non-conductor contacts positioned on a scarcely absorbent, reflective carrier characterised in that it comprises:
a microscope associated with a magnifying objective lens, and with image acquisition means to acquire a microscope image of the fluorescence of one of the contacts of the matrix or part of this contact;
illumination means emitting a first beam to allow image acquisition in white light of said contact, and a second beam to excite the fluorophor contained in said contact;
means for deflecting the second beam to ensure point by point scanning of the contact of the matrix;
means for recording the contact image and
means for processing this image to quantify the fluorescence of the contact and to determine the concentration of the substance mixed with the fluorophor, using fluorescence intensity, illumination intensity and integration time on the image acquisition means.
Advantageously, the illumination means comprise a ring illumination source which emits the first beam and a laser source which emits the second beam.
According to one embodiment of the invention, the image acquisition means comprise a cooled CCD camera, a colour filter placed in front of said camera and an electronic circuit ensuring the piloting of the photo multiplier and illumination means.
According to another embodiment of the invention, the image accusation means comprise a photo multiplier associated with an acquisition circuit, a colour filter placed in front of said photo multiplier and an electronic circuit ensuring the piloting of the camera and illumination means.
The microscope of the system of the invention my be fitted with a dichroic filter.
Advantageously, this microscope operates under epi-illumination.
Preferably, the deflector means of the system of the invention consist of an acoustic optical deflector deflecting the second beam in two orthogonal directions X and Y.
According to one embodiment of the invention, the laser is a micro laser; this may be a pulsated micro laser.
According to one variant of the invention, the colour filter is a high-pass filter.
In some applications, the matrix is an electrically-addressable matrix.
Also, the substance may be made up of DNA strands hybridised to probes made from synthesised DNA, and these DNA probes may be strongly fixed to the contacts with polypyrrol.
Depending upon the application, the fluorophor may be phycroerythrine, C-Snarf or sulphorhodamine, or any other fluorescent substance.
According to one embodiment, the system forms a reading box of small size and the contact matrix is carried on a card of xe2x80x9cchip cardxe2x80x9d type.
The invention also concerns the method of implementing the above system which is characterised in that it consists of:
a system calibration step with positioning and blocking of the matrix underneath the optical axis of the microscope objective, focusing the image acquisition means and automatic search of a reference contact in relation to the optical centre of the system;
an image acquisition step of one contact of the matrix; and
a calculation step of the fluorescence of said contact and subsequent deduction of the concentration of the substance.